Biological tests of blood plasma and other biological fluids frequently require that fluids be quickly divided into predetermined volumes for analysis in a variety of optical tests or assays. It is also frequently desirable to separate potentially interfering cellular components of the material from the other fluid prior to testing. Such measurement and separation steps have typically been performed by centrifugation to separate, for instance, blood plasma from the cellular components, followed by manual or automated pipetting of predetermined volumes of the blood plasma into separate test wells. Such procedures are labor intensive and time-consuming. As a result, various automated systems and methods have been proposed for providing multiple aliquots of plasma suitable for testing in a more efficient manner.
A major advance in the analysis of biological fluids has been the use of centrifugal rotors. These rotors are designed to measure volumes of a biological fluid, such as blood, remove cellular components, and mix the fluid with appropriate diluents for analysis, for example by optical testing. Typically, the rotors provide a plurality of samples in separate cuvettes in which the samples can be optically analyzed. Prior art rotors have frequently utilized complex designs which are costly and difficult to manufacture. Often, the rotors require various separable parts, which must be brought together or separated at different points in the centrifugation procedure. Previous centrifugal rotors have often been limited in the number of discrete samples and test wells they can provide. In some cases, these rotors require use of a separate displacement fluid to effect flow of blood and plasma through the system.
Many analytical devices exist employing centrifugal force to transfer fluids between assay chambers. U.S. Pat. No. 4,244,916 discloses a rotor comprising a plurality of cuvettes positioned radially outward of a central receptacle. Each cuvette is connected to the central receptacle by a duct and comprises a separate air escape orifice. U.S. Pat. No. 4,314,968 relates to rotors having cells positioned on the periphery of the rotor. Each cell includes a peripheral orifice for removing fluid introduced into the cell. U.S. Pat. No. 4,902,479 discloses a multi-cuvette rotor comprising elongated, radially extending cuvettes. Each elongated cuvette comprises a first chamber for receiving a first constituent and a second chamber for receiving a second constituent. A divider structure between the first and second chambers prevents mixing of the constituents before a predetermined time. Mixing occurs as the rotor is spun at a significant speed. U.S. Pat. No. 4,963,498 discloses devices relying on capillaries, chambers, and orifices to pump and mix fluids for optical analysis. U.S. Pat. No. 5,077,013 discloses rotors comprising peripheral cuvettes connected to holding chambers positioned radially inward from the cuvettes. U.S. Pat. No. 4,898,832 describes a rotor, which includes dried reagents adsorbed or bound to a solid carrier. A sample solution is moved along the rotor by use of centrifugal force and/or pressure force. U.S. Pat. No. 3,829,223 discloses a rotor adapted for mixing sample and reagent for photometric analysis in the rotor. Ramp-like projections on the walls of the test wells assist with mixing. U.S. Pat. No. 3,795,451 teaches a rotor for mixing a sample and reagent using a variation in rotational speed to provide mixing. A capillary passage is fed at increased rotational speeds to transfer the liquid as flow over a steep angle is permitted. U.S. Pat. No. 3,873,217 describes a rotor for photometric analysis using static loading of a main cavity and distribution of liquid to cuvettes using dynamic loading caused by rotational forces. U.S. Pat. No. 4,387,164 relates to chemical analyses of assay mediums and describes using reagents dispersed in soluble film. U.S. Pat. No. 3,881,827 teaches an apparatus and chamber for measuring cardiac output and includes a chamber for mixing a precise amount of dye with blood. U.S. Pat. No. 4,225,558 discloses a fluid test apparatus for multiple fluid samples. A sample and reagent are held in separate chambers until centrifugal force provides migration of the two fluids to a common chamber. U.S. Pat. No. 3,864,089 describes a rotor for blood fractionation. U.S. Pat. No. 4,509,856 is directed to a rotor useful for photometric analysis of a sample. U.S. Pat. No. 4,515,889 relates to the rotor having a plurality of interconnected small hollow spaces adapted for mixing reaction components. U.S. Pat. No. 4,689,203 relates to a centrifugal rotor designed for separating blood plasma from red and white blood cells. Although these inventions teach methods of combining, mixing and/or filtering samples and reagents in a rotary device, they do not allow automatic quantitative combination of reagent and sample in the device. The devices further do not provide precisely timed incubation of precisely combined reaction components before reaction products are transferred for detection.
For these reasons, it would be desirable to provide improved centrifugal rotors and methods suitable for quickly and easily mixing a volume of fluid with a reagent, and for transferring the resulting mixture from its mixing vessel to another chamber. It would be useful to transfer fluids into chambers suitable for separation of cellular components and ultimately distributed into test wells for analysis within the rotor.
U.S. Pat. No. 4,894,204 and U.S. Pat. No. 5,160,702 attempt to address some of these issues, but fail to provide high levels of reaction precision for large numbers of samples. Moreover, the complexity of design is problematic from a manufacturing standpoint. For example, the '702 patent discloses siphons for transferring fluids between chambers in a rotor. Calibration vessels have feed channels communicating with a central sample chamber and an exit orifice located in the wall opposite the feed channel. Although the siphon structures can meter somewhat controlled volumes, the precision is not high. Moreover, reaction timing and uniformity is compromised by the reaction chamber exit orifice designed to allow reaction product to escape while new fluids continue to enter. For higher precision, lower reagent and sample volumes and higher reproducibility, better fluid metering techniques would provide benefits.
U.S. Pat. No. 4,279,862 to Bretardiere is directed to a rotor, which has means for creating a pressure differential and/or turbulence to produce a homogeneous mixture of reagent and sample. Many assays can be preformed on the same assay rotor. However, sample and reagent volume precision requires manual pipetting of solutions into the device. Multistage reaction and detection schemes can not be run because reaction and detection must occur in the same chamber.
In view of the above, a need exists for analysis rotors able to accommodate relatively large numbers of test wells or cuvettes, and the rotor design should be simple and amenable to low-cost manufacture. It would be particularly desirable if the rotors were of unitary construction with no separable or movable parts. Liquid mixing methods should be simple and performable in relatively short times. Preferably, the assay methods should require relatively few steps and minimal human intervention. It would be advantageous if the methods required only rotation of the rotor in order to effect mixing and delivery of the fluid, e.g., at two or more chambers at different times. The present invention provides these and other features that will be apparent upon review of the following.